Alleles of imprinted genes are expressed differently depending on whether they are inherited from the male or female parent. Imprinting regulates a number of genes essential for normal development in mammals and angiosperms. In mammals, imprinted genes contribute to the control of fetal growth and placental development (Constancia, M. et al., Nature, 432, 53-57 (2004)). Human diseases are linked to mutations in imprinted genes or aberrant regulation of their expression (Constancia, M. et al., Nature, 432, 53-57 (2004)). Mechanisms of distinguishing maternal and paternal alleles have been extensively characterized in mammals. Imprinted genes reside in chromosomal clusters and are regulated by differentially methylated imprinting control regions (ICRs) (Reik, W. and Walter, J., Nat Rev Genet, 2, 21-32 (2001)). Differential DNA methylation is established during oogenesis or spermatogenesis by de novo methyltransferases and maintained somatically by the CG maintenance methyltransferase Dnmt1 (Li, E. (2002). Nat Rev Genet. 3, 662-673. ICRs are subject to differential histone modifications and in some instances can act as chromatin boundaries (Delaval, K. and Feil, R., Curr Opin Genet Dev., 14, 188-195 (2004)). Other mechanisms to regulate allele-specific gene expression involve non-coding RNAs, including antisense transcripts and microRNAs (O'Neill, 2005). Polycomb group (PcG) proteins, which function in large complexes to methylate histones and modify chromatin (Cao, R. and Zang, Y., Curr Opin Genet Dev., 14, 155-164 (2004)), maintain allele-specific silencing of some imprinted genes (Delaval, K. and Feil, R., Curr Opin Genet Dev., 14, 188-195 (2004)).
The endosperm, one of the products of angiosperm double fertilization, is an important site of imprinting in plants (Gehring, M. et al., Plant Cell, 16, S203-S213 (2004)) and has functions analogous to the placenta. In flowering plants, meiosis followed by mitosis produces the female and male gametophytes. Two cells of the female gametophyte, the haploid egg and the diploid central cell, are fertilized by two haploid sperm from the male gametophyte to form the diploid embryo and triploid endosperm, respectively. The endosperm provides nutrients to the embryo during seed development and, in Arabidopsis, is almost entirely consumed by the time embryo maturation is completed.
Molecular events that take place in the female gametophyte before fertilization have an essential role in endosperm gene imprinting. The imprinting of two genes, MEA and FWA, is regulated by DEMETER (DME, also sometime abbreviated DMT), a helix-hairpin-helix DNA glycosylase (Choi, Y. et al., Cell, 110, 33-42 (2002); Kinoshita, T. et al., Science, 303, 521-523 (2004)). DME has also been referred to in the literature as Atropos (ATR). The DME plant gene product has been described to control plant phenotypes and affect DNA methylation. The DME gene product is described in, e.g., U.S. Pat. Nos. 6,476,296 and 7,109,394 as well as Choi, Y. et al., Cell, 110:33-42 (2002); Gehring, M. et al., Cell, 124:495-506 (2006).
DNA glycosylases function in the base excision repair pathway by removing damaged or mismatched bases from DNA (Scharer, O. D. and Jiricny, J., BioEssays, 23, 270-281 (2001)). Bifunctional helix-hairpin-helix DNA glycosylases have both DNA glycosylase and apurinic/apyrimidinic (AP) lyase activities. The DNA glycosylase activity removes the damaged or mispaired base by cleaving the N-glycosylic bond, creating an abasic site, whereas the lyase activity nicks the DNA. An AP endonuclease generates a 3′-hydroxyl used by a DNA repair polymerase that inserts the proper nucleotide. A DNA ligase seals the nick to complete the repair process. DNA glycosylase/lyases have not been implicated in mammalian imprinting mechanisms.
Both MEA and FWA are expressed in the central cell before fertilization and in the endosperm, from the maternal allele, after fertilization (Kinoshita, T. et al., Science, 303, 521-523 (2004); Kinoshita, T. et al., Plant Cell, 11, 1945-1952 (2004); Vielle-Calzada, J. P. et al., Genes Dev, 13, 2971-2982 (1999)). In contrast, DME is expressed in the central cell of the female gametophyte but not in the endosperm (Choi, Y. et al., Cell, 110, 33-42 (2002)). Expression of MEA and FWA in the central cell and early endosperm is dependent on DME (Choi, Y. et al., Cell, 110, 33-42 (2002); Kinoshita, T. et al., Science, 303, 521-523 (2004)).
Though maternal expression of MEA and FWA is controlled by DME, there are important distinctions regarding the regulation of expression of these genes. FWA is silent in all vegetative and reproductive tissues except for expression of the maternal allele in the female gametophyte and endosperm (Kinoshita, T. et al., Science, 303, 521-523 (2004); Soppe, W. J. J. et al., Mol Cell, 6, 791-802 (2000)). MEA is imprinted in the endosperm, but is biallelically expressed in the embryo and in other sporophytic tissues (Kinoshita, T. et al., Science, 303, 521-523 (2004)). Expression of MEA in the embryo is likely not under DME control, as DME expression is not detected in the egg cell or embryo (Choi, Y. et al., Cell, 110, 33-42 (2002)). Expression of FWA in the endosperm, and elsewhere in the plant, is associated with hypomethylation of repeats in the 5′ region of the gene (Kinoshita, T. et al., Science, 303, 521-523 (2004); Soppe, W. J. J. et al., Mol Cell, 6, 791-802 (2000)). Paternal inheritance of met1 releases FWA paternal allele silencing in the endosperm and embryo (Kinoshita, T. et al., Science, 303, 521-523 (2004)). MET1 is the homolog of Dnmt1 (Bender, J., Ann Rev Plant Biology, 55, 41-68 (2004)).
DME, MEA, and MET1 genetically interact in the female gametophyte. MEA is an E(z) homologue that functions in a PcG complex along with FIE (Kohler, C. et al., EMBO J, 22, 4804-4814 (2003)), a homologue of Eed, to repress endosperm growth. Inheritance of mutant maternal dme or mea alleles causes endosperm overproliferation, embryo arrest, and seed abortion (Choi, Y. et al., Cell, 110, 33-42 (2002); Grossniklaus, U. et al., Science, 280, 446-450 (1998); Kiyosue, T. et al., Proc Natl Acad Sci USA, 96, 4186-4191 (1999); Luo, M. et al., Proc Natl Acad Sci USA, 96, 296-301 (1999)). Seed abortion caused by dme is suppressed by maternally inherited met1 if a wild type maternal MEA allele is present (Xiao, W. et al., Developmental Cell, 5, 891-901 (2003)). Moreover, met1 can restore MEA expression in dme mutants (Xiao, W. et al., Developmental Cell, 5, 891-901 (2003)). It is known that the glycosylase activity of DME is necessary for seed viability and activation of MEA transcription (Choi, Y. et al., Proc Natl Acad Sci USA, 101, 7481-7486 (2004)). DME antagonizes MET1 by specifically removing 5′-methylcytosine from MEA in the central cell, allowing the maternal MEA allele to be expressed there before fertilization and in the endosperm after fertilization.
As mentioned above, genetic information is stored not only in the sequential arrangement of four nucleotide bases, but also in covalent modification of selected bases (see, e.g., Robertson et al., Nature Rev. Genet. 1:11-19 (2000)). One of these covalent modifications is methylation of cytosine nucleotides, particularly cytosines adjacent to guanine nucleotides in “CpG” dinucleotides. Covalent addition of methyl groups to cytosine within CpG dinucleotides is catalyzed by proteins from the DNA methyltransferase (DNMT) family (Amir et al., Nature Genet. 23:185-88 (1999); Okano et al., Cell 99:247-57 (1999)). In the human genome, CpG dinucleotides are generally under represented, and many of the CpG dinucleotides occur in distinct areas called CpG islands. A large proportion of these CpG islands can be found in promoter regions of genes. The conversion of cytosine to 5′-methylcytosine in promoter associated CpG islands has been linked to changes in chromatin structure and often results in transcriptional silencing of the associated gene. Transcriptional silencing by DNA methylation has been linked to mammalian development, imprinting and X-Chromosome inactivation, suppression of parasitic DNA and numerous cancer types (see, e.g., Li et al., Cell 69:915-26 (1992); Okano et al., Cell 99:247-57 (1999)). Detected changes in the methylation status of DNA can serve as markers in the early detection of neoplastic events (Costello et al., Nature Genet. 24:132-38 (2000)).